發(fā)布時(shí)間：2018-05-07 08:17

  本文選題：甘草查爾酮B + 膀胱癌細(xì)胞��； 參考：《蘭州大學(xué)》2014年博士論文

【摘要】：目的：研究甘草查爾酮B(Licochalcone B,LCB)對(duì)膀胱腫瘤細(xì)胞生長(zhǎng)抑制及轉(zhuǎn)移能力的影響,并闡明其作用機(jī)制,為甘草查爾酮B治療膀胱腫瘤的臨床應(yīng)用提供理論依據(jù)。 方法：四甲基偶氮唑鹽實(shí)驗(yàn)(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide,MTT)測(cè)定甘草查爾酮B對(duì)T24、EJ細(xì)胞生長(zhǎng)的抑制效應(yīng)；Hoechst熒光染色觀(guān)察細(xì)胞形態(tài)變化；流式細(xì)胞技術(shù)檢測(cè)甘草查爾酮B對(duì)細(xì)胞周期和細(xì)胞凋亡率影響；實(shí)時(shí)定量PCR(Real-time quantitative PCR, qRT-PCR)檢測(cè)甘草查爾酮B對(duì)細(xì)胞周期相關(guān)蛋白在mRNA水平的影響；Westernblot檢測(cè)甘草查爾酮B對(duì)細(xì)胞分裂周期蛋白(CDC25A,CDC25B),以及凋亡相關(guān)蛋白Bcl-2,Bax,Caspase-3,PARP和Survivin表達(dá)的影響；采用MTT法檢測(cè)甘草查爾酮B對(duì)T24細(xì)胞與基質(zhì)粘附能力的影響；劃痕試驗(yàn)測(cè)定甘草查爾酮B對(duì)T24細(xì)胞體外遷移能力的影響；采用qRT-PCR.明膠酶譜兩種方法測(cè)定甘草查爾酮B對(duì)細(xì)胞基質(zhì)金屬蛋白酶(MMP-2,MMP-9)表達(dá)的影響；酶聯(lián)免疫法(ELISA)檢測(cè)甘草查爾酮B對(duì)激活蛋白-1(AP-1)、核轉(zhuǎn)錄因子(NF-κB)表達(dá)的影響。建立C57BL/6小鼠皮下膀胱癌模型,分離腫瘤組織,利用HE染色和Tunnel法檢測(cè)甘草查爾酮B對(duì)膀胱腫瘤的體內(nèi)抑瘤作用。 結(jié)果：(1)經(jīng)不同濃度(0,10,20,40,60,80μM)甘草查爾酮B分別處理24h,48h和72h,T24、EJ細(xì)胞增殖活力降低,當(dāng)甘草查爾酮B濃度增加到20μM以上時(shí)(40,60,80μM)細(xì)胞增殖率明顯低于對(duì)照組,呈濃度及時(shí)間依賴(lài)性變化；72h尤為明顯,ICso分別為39.18±0.51μM,34.15±0.36μM。 (2)T24和EJ細(xì)胞經(jīng)不同濃度甘草查爾酮B處理72h后,甘草查爾酮B能夠誘導(dǎo)腫瘤細(xì)胞S期阻滯；降低S期相關(guān)周期蛋白(cyclinA)及蛋白激酶(CDK1,CDK2)的mRNA表達(dá)；降低S期相關(guān)細(xì)胞分裂周期蛋白Cdc25A.Cdc25B在蛋白水平的表達(dá)。 (3)不同濃度甘草查爾酮B處理T24和EJ細(xì)胞72h后,能夠誘導(dǎo)細(xì)胞凋亡；下調(diào)Bcl-2表達(dá),上調(diào)Bax和Caspase-3在蛋白水平上的表達(dá),裂解PARP蛋白；并且在整體動(dòng)物水平上能夠抑制鼠膀胱腫瘤細(xì)胞增殖,誘導(dǎo)凋亡 (4)不同濃度甘草查爾酮B處理T24細(xì)胞24h后,細(xì)胞與基質(zhì)間粘附力降低；與對(duì)照組相比,甘草查爾酮B顯著降低MMP-9在mRNA及蛋白水平的表達(dá),但對(duì)MMP-2表達(dá)無(wú)明顯影響；能顯著抑制NF-1cB表達(dá),但對(duì)AP-1表達(dá)無(wú)明顯影響。 結(jié)論：以上研究結(jié)果說(shuō)明,甘草查爾酮B具有抑制膀胱腫瘤細(xì)胞生長(zhǎng)和轉(zhuǎn)移的作用,是一種有應(yīng)用前景的膀胱腫瘤灌注藥物。
[Abstract]:Objective: to study the effect of B(Licochalcone BB(Licochalcone on the growth inhibition and metastasis of bladder tumor cells, and to elucidate its mechanism, and provide theoretical basis for the clinical application of Glycyrrhiza chalcone B in the treatment of bladder cancer. Methods: the inhibitory effect of Glycyrrhiza B on the growth of T24EJ cells was determined by MTT assay. The morphological changes of T24EJ cells were observed by Hoechst fluorescence staining and the effects of glycyrrhizin B on cell cycle and apoptosis were detected by flow cytometry. The effect of glycyrrhizin B on cell cycle related protein (mRNA) was detected by real-time quantitative PCR(Real-time quantitative and qRT-PCR. Western blot was used to detect the effect of glycyrrhizin B on the expression of CDC25AnCDC25B, and apoptosis related protein Bcl-2nBaxCaspase-3PARP and Survivin. The effect of glycyrrhizin B on the adhesion of T24 cells to matrix was detected by MTT assay, the effect of glycyrrhizin B on the migration ability of T24 cells in vitro was determined by scratch test, and the migration ability of T24 cells was determined by qRT-PCR. The effect of glycyrrhizin B on the expression of matrix metalloproteinase-2 (MMP-2) MMP-9 and the expression of activator protein-1 AP-1 and nuclear transcription factor-NF-魏 B were detected by enzyme linked immunosorbent assay (Elisa). The subcutaneous bladder cancer model of C57BL/6 mice was established and tumor tissues were isolated. The inhibitory effect of glycyrrhizin B on bladder cancer in vivo was detected by HE staining and Tunnel method. Results (1) the proliferation activity of T24EJ cells treated with different concentrations of 10 ~ 10 ~ (20) and 40 ~ (6080 渭 M) glycyrrhizin B for 24 h and 72 h respectively decreased. When the concentration of glycyrrhizin B increased to more than 20 渭 M, the proliferative rate of T24EJ cells was significantly lower than that of the control group, and the proliferation rate of T24EJ cells was significantly lower than that of the control group. In a concentration and time dependent manner, ICso was found to be 39.18 鹵0.51 渭 m, 34.15 鹵0.36 渭 M for 72 h, respectively. After treated with different concentration of glycyrrhizin B for 72 hours, T24 and EJ cells could induce S phase arrest and decrease the mRNA expression of cyclin A and CDK1 + CDK2. The expression of cyclin Cdc25A.Cdc25B in S phase related cells was decreased at the protein level. T24 and EJ cells treated with different concentration of glycyrrhizin B for 72 h could induce apoptosis, down-regulate the expression of Bcl-2, up-regulate the expression of Bax and Caspase-3 at protein level, and break down the PARP protein. And it can inhibit the proliferation of bladder tumor cells and induce apoptosis on the whole animal level. (4) the adhesion of T24 cells treated with different concentration of glycyrrhizin B decreased after 24 hours, compared with the control group, glycyrrhizin B significantly decreased the expression of MMP-9 in mRNA and protein levels, but had no significant effect on the expression of MMP-2. It could significantly inhibit the expression of NF-1cB, but had no effect on the expression of AP-1. Conclusion: these results suggest that glycyrrhiza chalcone B can inhibit the growth and metastasis of bladder tumor cells and is a promising drug for bladder tumor perfusion.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.14

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